Open-channel block and unblock of voltage-gated Na channels, which results in resurgent Na current, is a specialized form of Na channel inactivation in many neurons. It increases channel availability and high frequency firing in cerebellar Purkinje cells. Upregulation or downregulation of the open channel blocking protein may change neuronal spiking, possibly providing a target for treatment of disease states that result from pathological firing patterns. The molecular mechanisms of this form of inactivation, however, have not been fully described. Knowledge of these mechanisms will increase understanding in the maintenance of activity patterns in cells expressing the specialized form of inactivation and may allow directed manipulation of activity in any neuron. Therefore, the major goals of this proposal are to define the mechanisms of open- channel block, specifically the identity of the blocker and whether it can be modulated in disease states. Specific aim one is to test the sufficiency of the Na channel beta4 subunit to produce block. Hippocampal CA3 pyramidal cells express the same alpha-subunits as Purkinje cells but do not have specialized inactivation kinetics, suggesting they do not contain the blocker (Raman and Bean, 1997). Based on the observation that the beta4 cytoplasmic tail peptide can mimic endogenous block (Grieco et al., 2005), expression of beta4 in CAS pyramidal neurons may be sufficient to produce resurgent current. To test this idea, the beta4 protein will be expressed in cultured CAS pyramidal cells. We will test for resurgent current by pulling nucleated patches from these cells and recording in voltage-clamp. The second aim of these experiments is based upon the observation that Na channel beta4 is significantly decreased in the cortex and basal ganglia in a model of Huntington's disease concurrently with symptom onset (Oyama et al., 2006). Thus, we will use these mice as a functional knockdown of beta4 to test if this protein can function as the blocking particle that produces resurgent current. We will first determine whether beta4 is reduced in cerebellar Purkinje cells using Western blotting and if so, acutely dissociate Purkinje cells and test for resurgent current by recording Na currents in voltage-clamp. We will also test whether some motor dysfunction of the disease can be attributed to the changes in resurgent current. Voltage-gated sodium channels are proteins responsible for the excitability of neurons in the central nervous system. Excitability is strongly limited by inactivation, a conformational change of these proteins. This project involves studying the molecular mechanisms of a form of Na channel inactivation, which could aid in the study of normal or pathological neuronal activity.